The long-term goal of this grant is to identify novel Hsp90 inhibitors with use in cancer therapy and in the treatment of neurodegenerative diseases. Several assays amenable for HTS that probe biochemically the inhibition of Hsp90 activity and in addition, cell-based assays that probe "fingerprints" of Hsp90 inactivation at cellular level have been developed by our laboratory. The goal of the current effort is to develop, optimize and validate our novel fluorescence Polarization assay for HTS utility. This assay probes the competitive binding of red-shifted BODIPY-labeled geldanamycin to Hsp90 (recombinant or from cell lysate).Here we aim to synthesize and characterize the red-shifted BODIPY-TMR-labeled geldanamycin ligand and use it in the optimization and validation of the competitive fluorescence polarization assay that probes forHsp90 inhibition by using either recombinant Hsp90 or Hsp90 from cellular lysates. We also aim to validate the assay for use in high-throughput screening. Running solely the FP binding assay will generate compounds that may be of high affinity but have poor cell permeability profiles. It could, also generate "false positives". We propose to validate hits by testing them in a cell based assay that reads a cellular fingerprint of Hsp90 inhibition. It was determined that addition of a Hsp90 inhibitor to SKBr3 cancer cells induced the rapid proteasomal degradation of the trans-membrane tyrosine kinase Her2, most protein being depleted at 6h. Thus we propose using our previously developed microtiter cell-based assay for the detection of agents that alter cellular levels of Her2 for this purpose. Hits resulted from the FP assay will be tested for Her2 degradation potency in the breast cancer cell line SKBr3. Highest ranking compounds in both screens will be taken for further testing in follow-up research programs. [unreadable] [unreadable]